Chromosomal rearrangements are .common in many types of human cancer and play an important role in carcinogenesis. However, the mechanisms of rearrangement in cancer cells remain poorly understood, thyroid cancer represents an excellent model to study these mechanisms because of a common occurrence of chromosomal rearrangements involving the RET gene, called RETIPTC, and well-established association with DMA damage induced by ionizing radiation. However, radiation exposure accounts only for a small portion of all thyroid tumors, offering a chance to study the role of other factors in the generation of rearrangements. The proposed study aims to test directly if DMA fragility participates in the generation of chromosomal rearrangements in human cells. Our preliminary results showed that fragile site-inducing chemicals can induce breaks in the RET gene, and the rearrangement can be generated under fragile-site inducing conditions. To further investigate this, .we will first examine whether one or more chromosomal regions involved in RETIPTC in thyroid cells are part of fragile sites. Specifically, we will determine directly the physical relationship between the fragile sites and the RET, H4, and ELE1 genes. We will also find the frequency of DMA breaks in these regions after treatment with fragile site-inducing chemicals. Then, we will test whether fragile site expression can lead directly to the generation of RETIPTC rearrangements in human thyroid cells. We will test directly the induction of RET/PTC1 and RET/PTC3 rearrangements in human HTori-3 thyroid cells after chemical induction of fragile sites, and after inactivation of ATR or BRCA1, which has been shown to increase fragile site expression dramatically. Finally, we will determine whether two characteristic features of fragile sites, late DNA replication and secondary structure formation, exist in the regions involved in RETIPTC rearrangements, contributing to the DNA breakage in these areas. These studies will determine whether fragile sites participate in the generation of RETIPTC rearrangement in thyroid cells and allow to explore the mechanisms of fragility in these regions. This will extend our understanding of the molecular mechanisms of chromosomal rearrangements in cancer cells, and may eventually lead to the development of novel therapeutic or preventive measures for malignant tumors developing thro.ugh the mechanism of chromosomal rearrangements.